The Banana MaWRKY18, MaWRKY45, MaWRKY60 and MaWRKY70 Genes Encode Functional Transcription Factors and Display Differential Expression in Response to Defense Phytohormones

WRKY transcription factors (TFs) play key roles in plant defense responses through phytohormone signaling pathways. However, their functions in tropical fruit crops, especially in banana, remain largely unknown. Several WRKY genes from the model plants rice (OsWRKY45) and Arabidopsis (AtWRKY18, AtWRKY60, AtWRKY70) have shown to be attractive TFs for engineering disease resistance. In this study, we isolated four banana cDNAs (MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70) with homology to these rice and Arabidopsis WRKY genes. The MaWRKY cDNAs were isolated from the wild banana Musa acuminata ssp. malaccensis, which is resistant to several diseases of this crop and is a progenitor of most banana cultivars. The deduced amino acid sequences of the four MaWRKY cDNAs revealed the presence of the conserved WRKY domain of ~60 amino acids and a zinc-finger motif at the N-terminus. Based on the number of WRKY repeats and the structure of the zinc-finger motif, MaWRKY18 and MaWRKY60 belong to group II of WRKY TFs, while MaWRKY45 and MaWRKY70 are members of group III. Their corresponding proteins were located in the nuclei of onion epidermal cells and were shown to be functional TFs in yeast cells. Moreover, expression analyses revealed that the majority of these MaWRKY genes were upregulated by salicylic acid (SA) or methyl jasmonate (MeJA) phytohormones, although the expression levels were relatively higher with MeJA treatment. The fact that most of these banana WRKY genes were upregulated by SA or MeJA, which are involved in systemic acquired resistance (SAR) or induced systemic resistance (ISR), respectively, make them interesting candidates for bioengineering broad-spectrum resistance in this crop.


Introduction
Banana is a staple food for more than 400 million people in the tropics and a major source of foreign currency for the leading producers [1]. Banana ranks as the most prolifically produced fruit in the world, with 145 million tons per year, representing an estimated value of USD$52 billion [2]. Similar to other monocultures, banana cultivars are affected by a broad range of pathogens, including viruses, bacteria, fungi, and nematodes, which can cause partial or total loss of fruit production [3,4]. Fungal pathogens are the most destructive in the banana industry today, and the most notorious of them are the resistance to the bacterial pathogens Erwinia carotovora and Pseudomonas syringae and to the fungal pathogen Erysiphe cichoracearum in transgenic Arabidopsis [22,23]. Both genes belong to group III. Another example is Arabidopsis AtWRKY18, whose overexpression increased resistance to P. syringae [24]. Moreover, Arabidopsis AtWRKY60 enhanced the DNA-binding activity of AtWRKY18 [25]. The AtWRKY18 and AtWRKY60 genes are phylogenetically closely related and belong to subgroup IIa of the WRKY family (Xu et al., 2006). Interestingly, homologs of AtWRKY18 and AtWRKY60 genes in banana were upregulated in response to the fungus P. fijiensis [26]. These reports and others make WRKY genes interesting candidates for the genetic improvement of crops.
In banana, genome-wide analyses have identified 164 members of the WRKY family, and transcriptomic analyses have revealed a possible involvement of members of groups II and III in disease resistance [26][27][28][29]. Although substantial advances have been made in the annotation of WRKY genes in banana and their expression analysis in response to biotic and abiotic stress, our understanding of their function as TFs and their regulation by defense hormones is still very limited. To expand the current knowledge of banana WRKY genes, in the present study, we isolated the cDNAs of four banana WRKY homologs, which we named MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70 due to their close phylogenetic relationships with their counterparts in Arabidopsis and rice. A comprehensive structural analysis of these banana WRKY genes was carried out, and their protein subcellular locations were determined. Furthermore, their function as TFs was determined, along with their expression profiles in response to the defense hormones SA and MeJA. The novel insights provided in this study represent a foundation for further functional analyses of these banana WRKY genes, which may lead to engineering disease resistance in this crop.

Plant Material and Phytohormone Treatments
Leaves of Musa acuminata ssp. malaccensis (ITC code: 1345) plants growing outdoors in leptosol soil at the Centro de Investigación Científica de Yucatán A. C. were used for RNA extraction and cloning of banana WRKY cDNAs. A floating leaf disc assay [30] was used for hormone treatments. Briefly, fully expanded young leaves next to the furled leaf of M. acuminata ssp. malaccensis adult plants were used to collect leaf fragments in the morning (9 a.m./26 • C), which were disinfected as described by Rodríguez-García et al. [31]. Leaf fragments were then cut into 10 circular pieces of 1.5 cm diameter and floated (adaxial surface facing up) on 30 mL of 5 mM SA [32] or 100 µM MeJA [33] in 10 cm diameter Petri dishes; both solutions contained 0.025% Silwet-L77 and 0.5% ethanol. For the control, the leaf discs were floated on 30 mL of 0.025% Silwet-L77 and 0.5% ethanol solution. All Petri dishes had 10 leaf discs, each excised from an independent plant. Three biological replicates were used for each hormone treatment, and three biological replicates were used for the control. Samples were incubated at 26 ± 1 • C in a growth room with a 16-h photoperiod (light intensity of 100 mmol m -2 s -1 ) for 6 h, and collected and stored at −80 • C until use for total RNA extraction.

BLAST Searches
The amino acid sequences of OsWRKY45 (GenBank accession number AK066255) [34] and AtWRKY70 (GenBank accession number AF421157) [22] from rice and Arabidopsis, respectively, were used as queries to search (E-value cutoff of −1) the proteome of the wild species M. acuminata ssp. malaccensis [26] using the BLASTP algorithm at the Phytozome v12.1 database (https://phytozome.jgi.doe.gov/pz/portal.html, accessed on 20 January 2021). We also included the banana WRKY sequences GSMUA_Achr3T13440 and GSMUA_Achr7T05200 as queries in BLASTP searches, as they have been shown to be upregulated by the pathogen P. fijiensis and have been reported to be closely related to AtWRKY18 and AtWRKY60 [26]. In this case, the BLASTP searches were carried out at the Phytozome v12.1 database (accessed on 20 January 2021) using the Arabidopsis proteome. The four banana WRKY genes were named based on their Arabidopsis or rice WRKY gene counterparts, with the prefix 'Ma' indicating M. acuminata.

RT-PCR and Cloning of MaWRKY cDNAs
The Illustra Nucleon PhytopureTM kit (GE Healthcare Life Science, Chicago, IL, USA) was used to extract total RNA from leaf tissue of the wild banana M. acuminata ssp. malaccensis following the manufacturer's instructions. Total RNA concentration and purity were determined using a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA integrity was determined by agarose gel electrophoresis stained with ethidium bromide. The nucleic acid extract was treated with TURBO™ DNase (Thermo Fisher Scientific, Waltham, MA, USA), and then first-strand cDNA synthesis was performed using 5 µg of total RNA employing SuperScript™ III (Thermo Fisher Scientific) according to the manufacturer's protocol. Specific primers for the predicted coding sequences (CDSs) of the MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70 genes were designed (Table S1) and used for RT-PCR amplification using the Expand™ Long Template PCR System (Merck, Darmstadt, Germany). The cycling conditions were 95 • C for 3 min, followed by 35 cycles of 95 • C for 10 s, 59 • C for 30 s, and 72 • C for 1 min. The PCR products were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) and sequenced on both strands by Macrogen, Inc. (https://dna.macrogen.Com, accessed on 8 February 2020). In addition, specific primers (Table S2) upstream of the stop codon of each CDS were designed for mapping the 3 cDNA ends using the SMARTer ® RACE Kit (Takara Bio USA, San Jose, CA, USA) according to the manufacturer's protocol. The PCR products were cloned and sequenced as described above. The sequences were edited using the BioEdit software v7.2. (Informer Technologies, Inc., Los Angeles, CA, USA) [35]. The cDNA sequences of MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70 were deposited in GenBank with the accession numbers OP186309, OP186310, OP186311, and OP186312, respectively.

Subcellular Localization Assay
The CDSs of the MaWRY18, MaWRY45, MaWRY60, and MaWRKY70 cDNAs were fused in frame to the 3 end of the GFP reporter gene present in the pAVA121 vector [40]. Onion epidermal cells were transiently transformed with the particle bombardment method [40] using a PDS-1000/He TM system (Bio-Rad, Hercules, CA, USA). Green fluorescence was observed with an AxioScope A1 epifluorescence microscope (Carl Zeiss, Oberkochen, Germany), and image editing was performed with ZEN Edition Blue imaging software (Carl Zeiss, Oberkochen, Germany).

Yeast One-Hybrid Assay
The transcriptional activity of MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70 was determined by yeast one-hybrid assay using the pGBKT7 vector and the Y2HGold yeast strain (Takara Bio USA, San Jose, CA, USA). The pGBKT7 plasmid contains the GAL4 DNAbinding domain (BD) under the control of the ADH1 promoter and the TRP1 nutritional marker for selection in yeast, whereas the yeast Y2HGold strain contains the reporter genes AUR1-C, ADE2, and HIS3. The growing conditions used for the yeast Y2HGold strain are described in the Matchmaker ® Gold Yeast Two-Hybrid System User Manual (Takara Bio USA). The CDSs of MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70 cDNAs were PCR amplified using primers designed for In-Fusion ® cloning (Table S1) and cloned into the pGBKT7 vector by recombination using the In-Fusion ® HD Cloning Plus kit (Takara Bio USA) following the manufacturer's instructions. The generated constructs were sequenced as described above and then used to transform the Y2HGold yeast strain, as described in the Matchmaker ® Gold Yeast Two-Hybrid System User Manual (Takara Bio USA). To test the transactivation activity, yeast strains harboring the recombinant and empty plasmids were streaked on synthetic dropout (SD) medium (-Trp, -His, -Ade) or YPDA medium supplemented with 200 ng/mL aureobasidin A. We used the papaya ERF transcription factor CpERF7 as a positive control [41].

RT-qPCR
Total RNA extraction and cDNA synthesis from floating leaf discs treated with SA or MeJA hormones were performed as previously described in Section 2.3. RT-qPCR was performed using Maxima SYBR Green/ROX qPCR master mix (Thermo Fisher Scientific) and specific primers for the MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70 genes ( Table S1). The cycling conditions were 95 • C for 10 min, followed by 40 cycles of 95 • C for 10 s, 57 • C for 30 s and 72 • C for 30 s. Banana Ma25S gene expression was used as an internal standard [42] to normalize the expression of the banana WRKY genes, and the 2 −∆∆Ct method [43] was used to calculate the relative gene expression. In addition, specific primers for the banana MaDLO1 [32] and MaLOX1 [33] genes were used in the RT-qPCR assays as controls (Table S1). RT-qPCR was carried out using the StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific). Three biological replicates (each one with three technical replicates) and two experimental repetitions were performed for statistical analysis. The Anderson-Darling statistic was used to determine whether the data meet the assumption of normality for a t-test, and then a Student's t-test was applied using the Minitab software v.17, (Informer Technologies, Inc., Los Angeles, CA, USA).

Structural and Phylogenetic
Analysis of the MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70 Genes BLASTP searches revealed that the best banana sequence hits to OsWRKY45 and AtWRKY70 were GSMUA_Achr5P07490 (E value = 1.78 × 10 −32 ) and GSMUA_Achr1P27980 (E value = 2.98 × 10 −25 ), respectively, whereas the best BLASTP hits for banana GSMUA_Achr3P13440 and GSMUA_Achr7P05200 in the Arabidopsis proteome were AtWRKY18 (E value = 1.81 × 10 −56 ) and AtWRKY60 (E value = 1.76 × 10 −58 ), respectively (Tables S3 and S4). The CDSs of the four banana WRKY cDNAs ranged from 828 (MaWRKY18) to 933 bp (MaWRKY70) in length (Table 1) (Figures S1-S4).  (Table S5). The deduced amino acid sequences of the four MaWRKY cDNAs revealed the presence of the conserved WRKY domain of~60 amino acids (Figures 1b and 2), which comprises the canonical WRKYGQK and zinc-finger motifs. Based on the number of WRKY repeats and the structure of the zinc-finger motif [18], MaWRKY18 and MaWRKY60 were determined to belong to group II, while MaWRKY45 and MaWRKY70 were classified as members of group III. Moreover, the four banana WRKY proteins had a potential nuclear localization signal (NLS) at the N-terminus near the WRKY domain ( Figure 1b). We also found potential phosphorylation sites in the four protein sequences (Figure 1b). All proteins except MaWRKY45 had posttranslational sites (PTSs) for myristoylation ( Figure 1b). Glycosylation sites were also found for MaWRKY18 and MaWRKY45 proteins (Figure 1b).

Subcellular Localization of MaWRK18, MaWRKY45, MaWRKY60, and MaWRKY70
As previously described, the four banana WRKY proteins were predicted to be located within the nucleus. To confirm whether these proteins were indeed localized in the nucleus of the cell, their coding sequences were fused in-frame to the GFP gene and transiently expressed in onion epidermal cells. We confirmed that these four MaWRKY proteins were localized inside the nucleus (Figure 6), indicating that these four MaWRKY proteins have roles in this cellular compartment.

Transactivation Activity of MaWRK18, MaWRKY45, MaWRKY60, and MaWRKY70 Transcription Factors
We carried out a yeast one-hybrid assay to test the in vivo transactivation activity of the MaWRKY18, MaWRKY45, MaWRKY60 and MaWRKY70 proteins. The cDNA coding sequences of these four MaWRKYs were fused in-frame to the GAL4 DNA-binding domain (Figure 7a), resulting in fusion proteins capable of activating the transcription of three independent reporter genes (AUR1-C, ADE2, and HIS3) and promoting the growth of the Y2HGold yeast strain in either SD minimal medium (-Trp, -His, -Ade) or YPDA medium supplemented with the highly toxic drug aureobasidin A (Figure 7b,c). The two negative controls used in the yeast one-hybrid assay did not grow on YPDA medium supplemented with aureobasidin A or SD (-Trp, -His, -Ade) minimal medium, whereas the positive control CpERF7 did grow (Figure 7c). These results indicated that these MaWRKY proteins were functional TFs capable of positively regulating the transcription process in yeast.

Subcellular Localization of MaWRK18, MaWRKY45, MaWRKY60, and MaWRKY70
As previously described, the four banana WRKY proteins were predicted to be located within the nucleus. To confirm whether these proteins were indeed localized in the nucleus of the cell, their coding sequences were fused in-frame to the GFP gene and transiently expressed in onion epidermal cells. We confirmed that these four MaWRKY proteins were localized inside the nucleus (Figure 6), indicating that these four MaWRKY proteins have roles in this cellular compartment.

Subcellular Localization of MaWRK18, MaWRKY45, MaWRKY60, and MaWRKY70
As previously described, the four banana WRKY proteins were predicted to be located within the nucleus. To confirm whether these proteins were indeed localized in the nucleus of the cell, their coding sequences were fused in-frame to the GFP gene and transiently expressed in onion epidermal cells. We confirmed that these four MaWRKY proteins were localized inside the nucleus (Figure 6), indicating that these four MaWRKY proteins have roles in this cellular compartment.  medium supplemented with the highly toxic drug aureobasidin A (Figure 7b, c). The two negative controls used in the yeast one-hybrid assay did not grow on YPDA medium sup plemented with aureobasidin A or SD (-Trp, -His, -Ade) minimal medium, whereas the positive control CpERF7 did grow (Figure 7c). These results indicated that these MaWRKY proteins were functional TFs capable of positively regulating the transcription process in yeast.

Expression Profiles of the MaWRK18, MaWRKY45, MaWRKY60, and MaWRKY70 Genes in Response to Phytohormone Treatments
To determine whether the MaWRK18, MaWRKY45, MaWRKY60, and MaWRKY70 genes were responsive to phytohormones involved in plant defense-signaling pathways we performed an RT-qPCR analysis to measure their transcript levels when exposed to exogenous SA or MeJA after 6 h. The four genes were responsive to SA (Figure 8). The expression levels of MaWRKY45 and MaWRKY60 increased above 2-fold, while To determine whether the MaWRK18, MaWRKY45, MaWRKY60, and MaWRKY70 genes were responsive to phytohormones involved in plant defense-signaling pathways, we performed an RT-qPCR analysis to measure their transcript levels when exposed to exogenous SA or MeJA after 6 h. The four genes were responsive to SA (Figure 8). The expression levels of MaWRKY45 and MaWRKY60 increased above 2-fold, while MaWRKY18 expression increased 1.7-fold. In the case of MaWRKY70, its expression decreased by 5-fold. The expression of the SA-responsive marker MaDLO1 increased significantly by 655-fold. The transcript levels in MeJA-treated samples exhibited higher expression levels than in samples treated with SA. The transcription levels of MaWRKY45, MaWRKY60, and MaWRKY70 increased significantly, with MaWRKY60 being the most responsive gene to MeJA treatment, with a 52-fold increase, whereas the MaWRKY18 gene was not responsive to MeJA. As an MeJA-responsive marker, the MaLOX1 gene was analyzed and showed a marked increase of 210-fold. These results suggest that the MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70 genes may play roles in SA or MeJA defense-signaling pathways. than in samples treated with SA. The transcription levels of MaWRKY45, MaWRKY60, and MaWRKY70 increased significantly, with MaWRKY60 being the most responsive gene to MeJA treatment, with a 52-fold increase, whereas the MaWRKY18 gene was not responsive to MeJA. As an ISR marker, the MaLOX1 gene was analyzed and showed a marked increase of 210-fold. These results suggest that the MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70 genes may play roles in SA or MeJA defense-signaling pathways.  , and MaWRKY70 genes in response to SA and MeJA phytohormones. A floating disc assay was performed in either SA (5 mM) or MeJA (100 µM) phytohormones. The leaf discs were incubated for 6 h and collected for total RNA extraction. Gene expression was evaluated by quantitative real-time PCR. The banana 25S gene was used as a reference gene for RT-qPCR analysis. The MaDLO1 and MaLOX1 genes were used as SA-and MeJA-responsive marker genes, respectively. The relative expression value was calculated by the 2 −∆∆CT method [43]. The mean ± S.D. of three biological replicates is presented. Asterisks (*) indicate p ≤ 0.05 (t test).

Discussion
The WRKY genes are widespread in plant genomes, where they play a fundamental role in the transcriptional regulation of genes involved in growth, development, and stress responses [45,46]. Annotation of WRKY genes in angiosperm plants has shown that they belong to a large gene family ranging from 38 to 287 members in Genlisea aurea and Glycine max, respectively [47]. Despite these advances in WRKY gene annotations, only a small percentage of them have been functionally characterized, mainly in the model plants Arabidopsis and rice. In the case of economically important fruit crops, such as banana, with 164 WRKY genes annotated [29], only a few WRKY genes have been validated as TFs [48][49][50][51]. Cloning and elucidating the functional aspects of banana WRKY genes will expand our understanding of the multiple roles of these transcription factors and provide novel tools for genetic improvement.
OsWRKY45, AtWRKY18, AtWRKY60, and AtWRKY70 play pivotal roles in the resistance to fungal and bacterial pathogens [22,25,34]. Given the importance of these TFs in plant defense responses, in this study, we searched the banana genome for potential orthologs of these genes. The MaWRKY45 and MaWRKY70 proteins resulted in the best BLASTP hits using their counterparts from rice and Arabidopsis as queries, respectively. It has previously been shown that OsWRKY45 and AtWRKY70 are phylogenetically closely related [52]; interestingly, MaWRKY45 and MaWRKY70 also clustered in the same phylogenetic clade as OsWRKY45 and AtWRKY70, suggesting an evolutionary relationship and function in stress responses. In the case of MaWRKY18 and MaWRKY60, their best BLASTP hits were with AtWRKY18 and AtWRKY60 from Arabidopsis, respectively. They shared the features of the group IIa WRKY TFs and clustered with AtWRKY18 and AtWRKY60, which are phylogenetically closely related [25], implying a possible orthology for MaWRKY18 and MaWRKY60.
We identified multiple potential phosphorylation sites in the protein sequences of MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70, even at the core of WRKY domains, which indicates that the activity of these banana WRKYs might be modulated via phosphorylation. Numerous WRKY TFs have been shown to be phosphorylated by mitogen-activated protein kinases, leading to enhanced binding to W-box elements and transcriptional activity [18]. We also identified N-myristoylation sites for these banana WRKY proteins. The number of characterized myristoylated proteins in plants is still limited [53], and to our knowledge, no myristoylated WRKY TF has been experimentally validated. Analysis of the myristoylome in Arabidopsis revealed that numerous membrane proteins involved in stress responses are myristoylated, suggesting that the myristoylome might function as a dynamic proteome in response to abiotic and biotic stresses [53]. Moreover, myristoylation of the BASP1 protein is required for its function as a transcriptional repressor by binding to the Wilms' tumor 1 (WT1) transcription factor and other corepressor proteins [54]. By analogy, some myristoylated MaWRKY TFs could work in a similar manner. We also found NLS sites in all four banana WRKY proteins considered in this study and confirmed that these proteins were indeed localized in the nuclei of onion epidermal cells. These results are consistent with the nuclear localization of other plant WRKY TFs, including the banana WRKYs reported thus far [48][49][50][51]55]. Furthermore, we demonstrated that these four banana WRKYs were functional transcription factors in yeast since all of them were capable of activating RNA polymerase II for the transcription of three marker genes. Less than 5% of the WRKY genes found in the banana genome have been experimentally validated as TFs, and most of them are from the cultivar Cavendish [48][49][50][51]; this is the first report that assessed the transactivation activity of WRKY proteins from a wild banana (M. acuminata ssp. malaccensis), which is resistant to the most destructive diseases of this crop and is a progenitor of most banana cultivars [26].
Numerous putative cis-acting regulatory elements related to defense phytohormones were found in the promoter regions of all four banana WRKY genes, including SA and MeJA, which are two key phytohormones involved in SAR and ISR, respectively [56]. MaWRKY18, MaWRKY45, and MaWRKY60 were upregulated in response to SA. This is in agreement with the expression profiles of AtWRKY18, OsWRKY45, and AtWRKY60, which were also upregulated by SA [24,34,57]. However, MaWRKY70 was downregulated by SA, in contrast to AtWRKY70, which has been shown to be upregulated by this phytohormone [22]. Regarding MeJa, MaWRKY45, MaWRKY60, and MaWRKY70 were upregulated in response to this phytohormone, unlike MaWRKY18, which was not responsive to MeJA. Interestingly, both MaWRKY18 and MaWRKY60 were upregulated by the fungal pathogen P. fijiensis in susceptible banana accessions [26]. Taking into consideration that MaWRKY18 was upregulated by SA and MaWRKY60 by both SA and MeJa, it is plausible that both phytohormones play roles in the banana-P. fijiensis pathosystem. In the case of Arabidopsis AtWRKY18 and AtWRKY60, it remains to be seen whether these TFs respond to MeJA. Previous reports have shown that OsWRKY45 and AtWRKY70 were upregulated by MeJa [22,57]. These two TFs function as a convergent point of jasmonate-and salicylate-mediated signals in plant defense [22,48]. Moreover, AtWRKY70 is regulated downstream of the master regulator of SAR, the transcription cofactor AtNPR1, while OsWRKY45 works independently of OsNPR1 [48], indicating that although these two TFs are phylogenetically closely related and function downstream of SA, they respond differently to NPR1. Thus, a similar scenario may occur in MaWRKY45 andMaWRKY70. Moreover, the promoter regions of MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70 contained W-boxes, suggesting that the expression of these banana WRKY genes could be either autoregulated or cross-regulated by other MaWRKYs, as previously reported [58]. W-boxes are ubiquitous in the promoter regions of stress-inducible genes [59,60].
The fact that these four MaWRKY genes were responsive to phytohormones involved in SAR and ISR makes them interesting candidates for engineering broad-spectrum resistance in banana. Since constitutive overexpression of WRKYs can lead to adverse effects on agronomic traits [21,24,34], it is necessary to fine-tune their expression through a moderate constitutive or pathogen-inducible promoter to harness the full potential of WRKY genes for boosting the plant immune system without detrimental effects on growth and development. Promising advances in this direction have been made in rice with a moderate constitutive and pathogen-inducible promoter driving the expression of OsWRKY45 [61,62]. Therefore, the use of these types of promoters in banana for driving the expression of MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70 may lead to triggering SAR or ISR, providing broad-spectrum resistance without affecting agronomic traits.

Conclusions
We isolated the cDNAs of four WRKY genes from a wild banana (M. acuminata ssp. malaccensis) and found that they belong to group II (MaWRKY18 and MaWRKY60) and group III (MaWRKY45 and MaWRKY70) of plant WRKY genes. Their proteins were located in the nuclei of onion epidermal cells and were functional TFs in yeast cells. Furthermore, these MaWRKY genes were responsive to the phytohormone SA, implying a possible role in SAR, while three of them were responsive to the phytohormone MeJA, suggesting a possible role in ISR. These results make MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70 interesting candidates for bioengineering broad-spectrum resistance in banana, using moderate constitutive or pathogen-inducible promoters to fine-tune their expression.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes13101891/s1, Table S1: List of primers for RT-PCR and RT-qPCR; Table S2: List of forward primers for 3 UTR amplification using RACE; Table S3: Best BLASTP hits of rice OsWRKY45 and Arabidopsis AtWRKY70 protein sequences used as queries in the proteome of M. acuminata ssp. Malaccensis; Table S4: Best BLASTP hits of banana GSMUA_Achr3P13440 and GSMUA_Achr7P05200 protein sequences used as queries in the Arabidopsis proteome; Table S5: Amino acid sequence similarity (%) between banana MaWRKY18, MaWRKY45, MaWRKY60, MaWRKY70 and their respective homologs in Arabidopsis and rice; Figure S1: cDNA sequence of MaWRKY18 and its deduced amino acid sequence; Figure S2: cDNA sequence of MaWRKY45 and its deduced amino acid sequence; Figure S3: cDNA sequence of MaWRKY60 and its deduced amino acid sequence; Figure S4: cDNA sequence of MaWRKY70 and its deduced amino acid sequence.